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. 2007 Mar;189(5):2046-54.
doi: 10.1128/JB.01375-06. Epub 2006 Dec 15.

Identification of IspC, an 86-kilodalton protein target of humoral immune response to infection with Listeria monocytogenes serotype 4b, as a novel surface autolysin

Linru Wang  1 , Min Lin
Affiliations

Identification of IspC, an 86-kilodalton protein target of humoral immune response to infection with Listeria monocytogenes serotype 4b, as a novel surface autolysin

Linru Wang et al. J Bacteriol. 2007 Mar.

Abstract

We identified and biochemically characterized a novel surface-localized autolysin from Listeria monocytogenes serotype 4b, an 86-kDa protein consisting of 774 amino acids and known from our previous studies as the target (designated IspC) of the humoral immune response to listerial infection. Recombinant IspC, expressed in Escherichia coli, was purified and used to raise specific rabbit polyclonal antibodies for protein characterization. The native IspC was detected in all growth phases at a relatively stable low level during a 22-h in vitro culture, although its gene was transiently transcribed only in the early exponential growth phase. This and our previous findings suggest that IspC is upregulated in vivo during infection. The protein was unevenly distributed in clusters on the cell surface, as shown by immunofluorescence and immunogold electron microscopy. The recombinant IspC was capable of hydrolyzing not only the cell walls of the gram-positive bacterium Micrococcus lysodeikticus and the gram-negative bacterium E. coli but also that of the IspC-producing strain of L. monocytogenes serotype 4b, indicating that it was an autolysin. The IspC autolysin exhibited peptidoglycan hydrolase activity over a broad pH range of between 3 and 9, with a pH optimum of 7.5 to 9. Analysis of various truncated forms of IspC for cell wall-hydrolyzing or -binding activity has defined two separate functional domains: the N-terminal catalytic domain (amino acids [aa] 1 to 197) responsible for the hydrolytic activity and the C-terminal domain (aa 198 to 774) made up of seven GW modules responsible for anchoring the protein to the cell wall. In contrast to the full-length IspC, the N-terminal catalytic domain showed hydrolytic activity at acidic pHs, with a pH optimum of between 4 and 6 and negligible activity at alkaline pHs. This suggests that the cell wall binding domain may be of importance in modulating the activity of the N-terminal hydrolase domain. Elucidation of the biochemical properties of IspC may have provided new insights into its biological function(s) and its role in pathogenesis.

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Figures

FIG. 1.
FIG. 1.
IspC and its deletion variants. (a) Polypeptides encoded by the expression constructs described in Materials and Methods. IspC, aa 1 to 774; EAD1, aa 58 to 197; EAD2, aa 58 to 263; EAD3, aa 1 to 197; CBD1, aa 198 to 774; CBD2, aa 234 to 774; CBD3, aa 249 to 774; CBD4, aa 264 to 774. (b to d) The purified recombinant IspC (1 μg) was analyzed by Western blotting, with probing with anti-His MAb at 0.1 μg/ml (b) and with rabbit antisera RαL (c) and RαK (d) at a dilution of 1:1,000. Protein standards (Std) with their molecular masses in kilodaltons are shown on the left of the blots. The arrow indicates the position of purified recombinant IspC.
FIG. 2.
FIG. 2.
Analysis of the peptidoglycan hydrolase activity of IspC by renaturing SDS-PAGE. The purified recombinant IspC (1.5 μg) was resolved on 12% gels containing 0.2% (wt/vol) autoclaved M. lysodeikticus ATCC 4698 (ML), 0.2% (wt/vol) autoclaved E. coli ATCC 25922 (EC), and 0.1% (wt/vol) autoclaved L. monocytogenes serotype 4b (LM) or on gels containing no bacteria (CB and WB). Hydrolysis of the peptidoglycan substrates occurred in 25 mM Tris-HCl (pH 7.5) containing 1% Triton X-100. ML, EC, and LM, peptidoglycan hydrolase activity staining; CB, Coomassie blue staining; WB, Western blot probed with anti-His MAb. The hydrolyase activity bands corresponding to the IspC protein band are indicated by an arrow.
FIG. 3.
FIG. 3.
Expression of the ispC gene in in vitro-cultured L. monocytogenes. The bacterial samples were taken at various time points (i.e., different OD620 values) over a 22-h growth period and analyzed for the ispC transcript by RT-PCR and for the protein by Western blotting, with probing with RαIspC at a dilution of 1:1,000. The RT-PCR products shown in each lane (i.e., at each time point) were derived from the same amount of total RNA (∼100 ng). For Western blot analysis, the whole-cell proteins from cells equivalent to 0.5 ml of culture at an OD620 of 0.5 at each time point and the purified rIspC (1 μg) were used. (a) PCR analysis of the ispC DNA without the reverse transcriptase step; (b) RT-PCR detection of the ispC mRNA; (c) PCR analysis of the 16S rRNA gene without the reverse transcriptase step; (d) RT-PCR detection of 16S rRNA as an internal control; (e) Western blot analysis of L. monocytogenes proteins and rIspC with the RαIspC antiserum.
FIG. 4.
FIG. 4.
Localization of IspC on the cell surface of L. monocytogenes serotype 4b by immunofluorescence staining. Bacterial cells (3 × 108) were probed with the RαIspC antiserum (a) or preimmume serum (c), followed by reaction with FITC-conjugated goat anti-rabbit antibody, as described in Materials and Methods. Cells were visualized with a fluorescence microscope. Fluorescence images (a and c) and phase-contrast images (b and d) of the bacterial cells in the same field are shown.
FIG. 5.
FIG. 5.
Localization of IspC on the cell surface of L. monocytogenes serotype 4b by immunogold labeling. Bacterial cells (3 × 108) were probed with the RαIspC antiserum (a) or preimmume serum (b), followed by interaction with gold (12 nm)-conjugated goat anti-rabbit IgG (heavy plus light chains), as described in Materials and Methods. Bacterial cells were visualized with a transmission electron microscope at a magnification of ×30,000, showing gold particles on the surface. Bar, 0.6 μm.
FIG. 6.
FIG. 6.
Binding of the IspC C-terminal regions fused with GFPuv to the cell surface of L. monocytogenes serotype 4b. Live L. monocytogenes (6 × 108 cells), harvested from the late exponential growth phase, was incubated with GFPuv-CBD fusions or GFPuv, each at 0.564 nM, at room temperature for 5 min and imaged with a fluorescence microscope (left panels). The C-terminal regions of IspC are CBD1 (aa 198 to 774), CBD2 (aa 234 to 774), CBD3 (aa 249 to 774), and CBD4 (aa 264 to 774). The exposure times for capturing images are indicted by numbers in parentheses. The right panels show corresponding phase-contrast images of the bacterial cells in the same field.
FIG. 7.
FIG. 7.
Peptidoglycan hydrolase activity of the full-length recombinant IspC as a function of pH. Renaturing SDS-PAGE was performed using M. lysodeikticus ATCC 4698 substrate (0.2% wt/vol) as described in the legend to Fig. 2. Sodium citrate-citric acid (pH 3-6), Tris-HCl (pH 7.5-9.0), and glycine-NaOH (pH 10) buffers were used.
FIG. 8.
FIG. 8.
Mapping of a peptidoglycan hydrolase domain in the N-terminal region of IspC. Renaturing SDS-PAGE analysis of the purified recombinant proteins derived from the N-terminal region (lanes 1, EAD1 [aa 58 to 197]; lanes 2, EAD2 [aa 58 to 263]; lanes 3, EAD3 [aa 1 to 197]) was performed using the M. lysodeikticus ATCC 4698 substrate (0.2%, wt/vol) at various pHs. The same buffers as described in the legend to Fig. 7 were used. The positions to which the purified EADs migrated were shown by Western blotting (WB), with probing with an anti-His MAb.
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References

    1. Baba, T., and O. Schneewind. 1996. Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus. EMBO J. 15:4789-4797. - PMC - PubMed
    1. Baba, T., and O. Schneewind. 1998. Targeting of muralytic enzymes to the cell division site of Gram-positive bacteria: repeat domains direct autolysin to the equatorial surface ring of Staphylococcus aureus. EMBO J. 17:4639-4646. - PMC - PubMed
    1. Berry, A. M., and J. C. Paton. 2000. Additive attenuation of virulence of Streptococcus pneumoniae by mutation of the genes encoding pneumolysin and other putative pneumococcal virulence proteins. Infect. Immun. 68:133-140. - PMC - PubMed
    1. Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-254. - PubMed
    1. Braun, L., S. Dramsi, P. Dehoux, H. Bierne, G. Lindahl, and P. Cossart. 1997. InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association. Mol. Microbiol. 25:285-294. - PubMed

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